Cloning And Functional Study Of Genes Involved In Promoting Plant Growth Using A Bacillus Subtilis OKB105 Tnylb-1 Transposition Insertion Library

Bacillus subtilis, as an important type of plant growth-promoting rhizobacteria (PGPR), not only has broad-spectrum antimicrobial activity and stress resistance, but also can significantly promote plant growth by root application. Although the advantages of plant disease biocontrol by B. subtilis is more and more obvious now, the molecular mechanisms of colonizing in plant rhizosphere and promoting plant growth of B. subtilis have not been understood. This research was aimed at identifying the genes involved in promoting plant growth using the Bacillus subtilis OKB105 mutant library and studying the functions of these relative genes.At present, the ability to create random DNA insertions in bacterial chromosomes has been a very powerful technique for discovering new genes and discovering new functions of known genes. The transposable element Himarl, a member of the mariner family of transposons, has been extensively used to generate libraries of mutants in many bacteria. When compared to other transposons, Himarl transposon has been shown to insert randomly into the chromosomes of a number of bacterial species with high transposition frequency, and has a broad host range. B. subtilis OKB105, a derivative strain of B. subtilis 168, can protect plants against CMV and TMV, and significantly enhanced the plant height and fresh weight by enhancing the expression of plant growth-related genes, In this study, insertional mutagenesis of B. subtilis OKB105 was performed by use of a Himarl-based transposon TnYLB-1, and 2000 transposants were selected randomly. PCR and southern hybridization analyses of 15 transposants showed that the plasmid pMarA carrying the transposon TnYLB-1 could transform into OKB105 successfully, and TnYLB-1 transposed into the B. subtilis chromosome randomly under the condition of high temperature.7 mutants defective in promoting plant growth were selected from the Bacillus subtilis OKB105 TnYLB-1 mutant library. The results of square Petri dishes experiments demonstrated that the supernatant liquid of B. subtilis OKB105 could significantly promote the growth of tobacco roots, the root growth had been increasd by 55.9%. In contrary, supernatant liquid of the 7 mutants did not have a positive effect on the root growth of tobacco. To identify the genes in these mutant loci, the inserted transposon and its flanking regions were cloned by inverse PCR and sequenced from each mutant. Transposon insertions in these seven mutants were found to be in the gene trxB, pycA, yfnl, ybfQ, rsmG, ycdG, lytB. The functions of these genes are respectively relative to encoding the proteins such as thioredoxin reductase, pyruvate carboxylase, transmembrane glycoprotein, enzyme with rhodanese domain, glucose-inhibited division protein, modifier protein of major autolysin LytC, oligo-1,6-glucosidase.These genes may have effects on biological function of cell membrane and glucose metabolism of B. subtilis.M3, a mutant defective in promoting plant growth, was obtained by screening the TnYLB-1 transposon mutant library of B. subtilis OKB105. Inverse PCR demonstrated that the insertion site of M3 was gene yfnI. In this study, a shuttle vector pMK4-yfnI was constructed and transformed into M3 to restore the function of yfnl gene. The complementation experiment showed that the transformant M3ΔyfnI had the same effect with OKB105 on promoting the growth of tobacco roots, the root growth had been increasd by 65%. The results verified that yfnl was significantly correlated with promoting plant growth of B. subtilis OKB105. The result of growth curve determination of M3 and OKB105 showed that M3 had the same growth ability with OKB105 in both LB medium and Landy medium. It was concluded that the growth of B. subtilis OKB105 was not be affected if yfnl had been mutated. In the present study, there are there genes alsD, yhcX and ysnE relative to promoting plant growth of B. subtilis OKB105. Quantative RT-PCR analysis demonstrated that in M3 the expression level of these three genes showed no significant change compared to wild type OKB105, confirming that the disrupted gene has no effect on the transcription of the three genes. Finally, it was assumed that gene yfnl could regulate the ability of promoting plant growth of B. subtilis OKB 105 according to an unknown mechanism.